The broad objective of this project is to better understand tRNA function, regulation, and the dynamic contributions of individual tRNAs within the cell. Given the rapidly growing collection of genomic data and high throughput RNA detection methods, there are new opportunities to advance tRNA research if known limitations in our computational and molecular detection methods are overcome. Thus, the aims of this grant are: (1) To expand and improve the technology of computational tRNA detection, (2) To provide a comprehensive database and advanced analysis tools enabling new biological insights and experimentation by the tRNA research community, (3) To measure the complex dynamics of tRNA abundance to understand the regulation and cellular roles of individual tRNA gene loci (4) To identify and test the biological contribution of atypical but evolutionarily recurrent tRNAs which challenge our conventional understanding of tRNA sequence and structure To achieve these aims, we will develop improved tRNA detection methods based on a greatly expanded sampling of tRNA diversity, with new, more specialized probabilistic search models. With major improvements in the design, content, and function of the Genomic tRNA Database, we will provide the most complete and up-to-date collection of tRNAs and on-line tools for tRNA researchers. To link expression data to specific tRNA loci, we will collect a wide phylogenetic sampling of tRNA-seq data, utilizing specialized mapping and normalization methods. To better understand what distinguishes a functional tRNA from a pseudogene, we will use comparative genomics and traditional biochemistry to assay function of a diverse set of natural tRNA variants in yeast. These tools, data resources, and studies will enable and accelerate biomedical research in the tRNA community. tRNA researches study a wide variety of human diseases that can be caused by mutations in mitochondrial tRNAs including diabetes, blindness, myoclonic epilepsy, cardiomyopathy, Alzheimer's Disease, Parkinson's Disease, and neurosensory hearing loss. Because increased tRNA abundance is required for rapid cell division in tumorigenesis, identification of disregulated tRNA loci could lead to new targets for cancer therapies. Finally, processing, editing, and modification of tRNAs differ in many ways between bacteria and eukaryotes - closer study of these phylogenetic differences could offer opportunities to develop novel antibiotics based on pathogen-specific tRNA processing enzymes.